The aim of this project is to uncover the mechanism of action of nerve growth factor (NGF), with particular emphasis on NGF-promoted initiation, locomotion, maintenance and regeneration of neurites. Experiments will principally employ cultured sympathetic neurons and the NGF-responsive PC12 pheochromocytoma line developed in this laboratory. The proposed investigation will draw heavily upon existing dat produced under this grant. The following four major separable, but integrated subsets of experimnal topics shall be pursued. I. The transductional mechanisms of NGF. The means by which binding of nGF to its receptors triggers subsequent actionof the factor will be studied. The roles of phosphorylation, intracellular Ca++ activity and methylation will be explored in particular. II. Short-latency, local transcription-independent actions of NGF. This will include the rapid activation of tyrosine hydroxylase in response to NGF and the underlying role of phosphorylation in this phenomenon. Also in this subset, video time lapse recording, scanning EM and biochemical analyses will be employed to describe and mechanistically probe the local regulation by NGF of growth cone shape, motility and locomotion. III. Delayed, transcription-dependent actions of NGF. The induction of microtubule-associated proteins (MAPs) by NGF will be further investigated as well as the role of these proteins in the mechanism by which NGF promotes neurite outgrowth. IV. Genetic analysis of the NGF mechanism. The PC12 line is especially well suited to such an approach. Mutants fo PC12 cells deficient in specific responses to NGF (especially neurite outgrowth) will be generated, selected and characterized. These will be exploited so as to ultimately uncover the identities and functional roles of each of the gene products involved in NGF's actions. As a whole, the above studies relate to the basic mechanisms by which nerves are stimulated to grow and regenerate and have relevance to a number of issues in cellular neurobiology.